International Journal of Antimicrobial Agents 22 (2003) 301 /317 www.ischemo.org
ABC transporters and drug resistance in parasitic protozoa Antonios Klokouzas 1, Sanjay Shahi 1, Stephen B. Hladky, Margery A. Barrand, Hendrik W. van Veen * Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK
Abstract Parasitic protozoa are responsible for a wide spectrum of diseases in humans and domestic animals. The main line of defence available against these organisms is chemotherapy. However, the application of chemotherapeutic drugs has resulted in the development of resistance mechanisms, which limit the number of antiprotozoal drugs that are effective in the treatment and control of parasitic diseases. Knowledge about the resistance mechanisms involved may allow the development of new drugs that minimise or circumvent drug resistance or may identify new targets for drug development. This review focuses on the role of protozoal ATPbinding cassette (ABC) transporters in drug resistance. These membrane proteins mediate the ATP-dependent transport of a wide variety of chemotherapeutic drugs away from their targets inside the parasites. The genome sequence of Plasmodium falciparum and Plasmodium yoelii has recently been completed, and the sequencing of other parasitic genomes are now underway. As a result, many new membrane transporters belonging to the ABC superfamily are being discovered. We review the ABC transporters in major parasitic protozoa, including Plasmodium , Leishmania , Trypanosoma and Entamoeba species. Transporters with an established role in drug resistance have been emphasised, but newly discovered transporters with a significant amino acid sequence identity to established ABC drug transporters have also been included. # 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: ABC transporters; Drug resistance; Protozoa
1. Introduction Diseases caused by parasitic protozoa are a major cause of mortality and morbidity throughout the world. Malaria is by far the world’s most important parasitic disease, and kills more people (2 /3 million) than any other communicable disease except tuberculosis (World Health Organisation, WHO) [1,2]. Other life-threatening protozoal infections include African sleeping sickness, Chagas disease, toxoplasmosis, amoebic dysentery and cryptosporidiosis (WHO) [3]. Some of the parasitic infections, such as leishmaniasis and cryptosporidiosis, have emerged as HIV-coinfections [4,5]. In addition, non-life-threatening protozoal diseases caused by Giardia duodenalis (diarrhoea) and Trichomonas vaginalis (urethritis, vaginitis) are widespread. The prevalence and geographical distribution of the major parasitic protozoal infections are described in Table 1.
* Corresponding author. Fax: /44-1223-33-4040. E-mail address:
[email protected] (H.W. van Veen). 1 These authors contributed equally.
Because prevention of parasitic infections by vaccination has up to now been unsuccessful [6,7], control of infections has been largely through the use of chemotherapeutic drugs that are cytotoxic to the parasitic microorganisms. However, in the past decade a major obstacle to chemotherapy has been the emergence of drug resistance by these organisms [8 /11]. An increasing number of Leishmania species have become resistant to first-line drugs (e.g. pentavalent antimonials) [12]; Trypanosoma brucei , the causative agent of African sleeping sickness, has become resistant to the clinical drugs melarsoprol and diamidines [13]; Plasmodium falciparum , which is responsible for the most dangerous form of malaria, now shows resistance to antifolates and chloroquine (CQ) [10]. Other pathogenic protozoa, such as Trypanosoma cruzi , Giardia lamblia , T. vaginalis , and Entamoeba histolytica have also developed a clinically significant drug resistance [14 /16]. At present much scientific effort is spent on elucidating the mechanism(s) underlying this resistance with the hope of improving the efficacy of existing drugs and of developing new drugs that can bypass the resistance mechanisms.
0924-8579/03/$30 # 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/S0924-8579(03)00210-3
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Table 1 Major diseases caused by parasitic protozoa in humans Disease
Causative organism
Vector/intermediate Endemic region(s) host
Prevalence (in millions)
Malaria
Plasmodium spp.
300 /500
Leishmaniasis
Leishmania spp.
Anopheline mosquito Phlebotomine sandfly Tsetse fly (glossina )
Central and South America Middle 10 /15 East, India, China Sub-Saharan Africa 0.3 /0.5
Triatomine insect
Mexico to South Argentina
African sleeping sickness Trypanosoma brucei gambiense , Trypanosoma brucei rhodesiense South-American ChaTrypanosoma cruzi gas’ disease
Amongst the various mechanisms identified, those based on drug transport appear to play an important role by relocating the drugs away from their target sites within the parasites. Transporters of the ATP-Binding Cassette (ABC) family are known to provide the basis of multidrug resistance in mammalian cancer cells and in pathogenic yeasts, fungi and bacteria [17 /22]. These proteins constitute one of the largest families of membrane proteins found in both pro- and eukaryotes. In general they are composed of at least four domains: two membrane domains (MDs), each usually with six putative a-helical transmembrane segments (TMS), and two nucleotide-binding domains (NBDs). The NBDs contain the ‘‘Walker A’’ and ‘‘Walker B’’ motifs [23] common to all ATP-binding proteins. In addition, the NBDs possess a short, highly conserved sequence just upstream of the Walker B site called the ‘‘ABC signature’’ which is typical for the members of the ABC family (Fig. 1) [24].
Sub-Saharan Africa
16 /18
Some of the best known ABC transporters associated with clinical resistance in humans are P-glycoprotein (Pgp) encoded by the MDR1 gene and certain members of the subgroup of Multidrug Resistance associated Proteins encoded by genes, MRP1-8 . The breast cancer resistance protein (BCRP) is another more recently discovered human multidrug transporter that belongs to the ABC family. These transporters have become targets for inhibition by agents used alongside anticancer drugs as ‘resistance modulators’ to enhance chemosensitivity of resistant tumours. Modulators for Pgp include desipramine and verapamil. The idea that ABC transporters might also be involved in resistance to antiprotozoal drugs has come from initial findings that (i) the antimalarial drug CQ accumulates less in CQresistant P. falciparum than in CQ-sensitive parasites as a result of enhanced drug extrusion [25] (ii) CQ resistance in P. falciparum appears to be modulated in vitro by the human Pgp modulator verapamil [25], and
Fig. 1. (A) Schematic diagram of a typical ABC transporter containing two MDs and two NBDs. (B) Characteristic motifs (represented by shaded rectangles) in the NBDs of ABC transporters. The consensus sequences of the Walker A, Walker B, and ABC signature motifs are given at the bottom with invariant residues shown in capital letters and the conserved residues in lower case letters. The consensus sequences were adapted from Saurin [135]. IN and OUT refer to the inside and outside of the membrane, respectively.
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(iii) arsenite efflux in Leishmania is reversed by desipramines and verapamil. These observations prompted the search for ABC-type MDR transporters in parasitic protozoa [26 /29]. At least 20 ABC (multi) drug transporter have now been identified in parasitic organisms. This number may increase rapidly with the identification of new ABC transporter genes in ongoing genome sequencing projects (see http://www.ebi.ac.uk/parasites/parasite-genome.html for up-to-date information). Members of the ABC transporter superfamily in human, yeast, plants and bacteria have been subdivided into various clusters based on the extent of their sequence homology. Here, we present such an analysis for parasitic protozoa, and we discuss the role of selected transporters in the drug resistance of the protozoal parasites Leishmania , Trypanosoma and Plasmodium .
2. Phylogenetic analysis of ABC transporters in parasitic protozoa Table 2 provides an overview of all known ABC transporters that have been identified in parasitic protozoa to date. Many of the genes encoding these proteins were isolated primarily by the approach of cloning homologous genes using PCR and degenerate primers directed against the conserved NBD domains of ABC transporter genes from other organisms (see Fig. 1). Other approaches have included hybridisation of genomic libraries, and inspection of DNA sequence databases. The latter approach has now become very efficient as genome sequence programs are under way for various parasitic protozoans. In addition to the ABC transporters with an established role in drug resistance, new ABC transporters have been identified for which a role in drug resistance has not yet been shown. In order to analyse the phylogenetic relationship of ABC transporters in parasitic protozoa, the individual amino acid sequences corresponding to the MDs (Fig. 2A) and NBDs (Fig. 2B) in the transporters were compared and aligned, and unrooted phylogenetic trees were constructed based on the percentages of identical amino acids found for each pair of sequences in the alignment. The phylogenetic tree for the NBDs displays a Pgp cluster and separate MRP clusters for the N- and C-terminal domains. The tree for the MDs also displays clear N- and C-terminal MRP clusters, but the remaining sequences are not clearly grouped by this fairly primitive level of analysis. These results suggest that the MDs and NBDs of the MRPs have co-evolved from ancestral ABC transporter genes in which MD and NBD coding regions were already fused into halftransporters. The Pgp cluster contains protozoal ABC transporter homologues of the human Pgp, such as Pf MDR1, Le MDR1 and Eh PGP1 containing two fused
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half-transporters each composed of an amino-terminal MD followed by a carboxy-terminal NBD [referred to as an {MD-NBD}2 topological configuration]. The Pgp cluster also encompasses parasitic half-transporter proteins with a {MD-NBD} topology such as Tv PGP1 and Pf MDR2. Two hypothetical ABC proteins CAB10570 and AAF9945 from P. falciparum and P. vivax, also have the {MD-NBD} topology (Fig. 3). Interestingly, no evidence has yet been found for the presence of parasitic half-transporters similar to the human BCRP with a reversed {NBD-MD} topology. The MRP cluster contains protozoal homologues of human MRP proteins such as Lt PGPA, Cp ABC or Tc PGP2. They share the {MD-NBD}2 topology with members of the Pgp cluster, but with either a unique long (/200 residues) hydrophilic amino-terminal sequence as observed in human MRP4 and MRP5, or an additional hydrophobic MD0 domain as observed in human MRP1. The MRP cluster also includes a new putative ABC transporter, termed CAB64568 from L. major (Fig. 2). In the following sections, the molecular properties of protozoal members of the Pgp and MRP clusters will be discussed in more detail.
3. Protozoal ABC transporters found in the Pgp cluster Pgp-related genes with significant homology to the mammalian MDR1 gene have been described in Leishmania. The first MDR1 gene homologue in this organism was amplified on an extrachromosomal circle in a stepwise selected vinblastine-resistant L. donovani [30,31]. Homologues of the LdMDR1 gene have also been described in vinblastine-resistant L. enrietti (LeMDR1 ) and L. mexicana (LmMDR1 ) and in daunorubicin-resistant L. tropica (LtrMDR1 ) and L. amazonensis (LaMDR1) [32 /35]. Six different Pgp homologues have been identified in emetine-resistant E. histolytica : four complete genes (EhPGP1, EhPGP2, EhPGP5 , and EhPGP6) and two pseudogenes (EhPGP3 and EhPGP4 ) that contain stop codons in the coding sequence [36 /39]. The open reading frames of the complete genes show 38/41% identity to mammalian MDR1 and 22/27% and 11/ 18% to those of Plasmodium and Leishmania , respectively. Gene transfection experiments have confirmed that overexpression of Eh PGP1 confers emetine-resistance in E. histolytica [40]. A typical half-transporter homologue of mammalian Pgp has been described in the flagellated protozoan T. vaginalis , the causative agent of vaginitis and urethritis [41]. The gene, termed TvPGP1, encodes a protein with a N-terminal MD composed of six TMS, and a Cterminal NBD. Overexpression but no amplification of TvPGP1 gene has been reported in metronidazoleresistant strains of T. vaginalis [41].
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Table 2 ABC transporters in parasitic protozoa Organism
Plasmodium spp. P. falciparum c
ABC gene
PfMDR1 (chr 5)
Leishmania spp. L. tarentolae
L. donovani L. amazonensis L. major
L. enriettii L. tropica Trypanosoma spp. Trypanosoma brucei
Trypanosoma cruzi Cryptosporidium parvum Entamoeba histolytica
Trichomonas vaginalis
NBD Drug resistance a
GenBank accession num- References ber
2
Yes
M29154
[26,27]
b
Pf MDR1/Pgh1 (1419 aa) Pf MDR2/Pgh2 (947 aa;1025 aa) Pf GCN20 (816 aa) CAB63558 (1822 aa)
1
No (?)
L13381, U04640
[45 /47]
2 2
? ?
U37225 AL031746
[61]
CAB10570 (1365 aa)
1
?
Z97348
AAN36368 (2108 aa)
2
?
AE014848
AAF99456 (1364 aa)
1
?
AY003872
LtPGPA LtPGPB LtPGPE LdMDR1 LaMDR1 gij9557975:224 680 / 228 373d gij6635092:1 /4716d LeMDR1 LtrMDR1 LtrPGPE
Lt PGPA (1548 aa) Lt PGPB (1451 aa) Lt PGPE (1724 aa) Ld MDR1 (1341 aa) La MDR1 (1341 aa) CAC00243 (1231 aa)
2 2 2 2 2 1
Yes No (?) No (?) Yes Yes ?
X17154 L29484 L29485 L01572 AB003329 AL160371
CAB64568 (1571 aa) Le MDR1 (1280 aa) Ltr MDR1 (1341 aa) Ltr PGPE (1677 aa)
2 2 2 2
? Yes Yes ?
AL135898 L08091 U63320 U55381
[130] Unpublishede Unpublishede
TbMRPA TbMRPE TbABC2 f TbABC3 f TcPGP1A TcPGP2 CpABC
Tb MRPA (1581 aa) Tb MRPE (1759 aa) Tb ABC2 Tb ABC3 Tc PGP1A (1034 aa) Tc PGP2 (1534 aa) Cp ABC (1431 aa)
2 2 1? 1? 1 2 2
Yes Yes ? ? ? No (?) ?
AJ318885 AJ318886 U89027 U89028 U95956 Z49222 AF110147
[43] [43] [42] [42] [60] [59] [55]
EhPGP1 EhPGP2 EhPGP5 EhPGP6 EhABC1 TvPGP1
Eh PGP1 (1302 aa) Eh PGP2 (1310 aa) Eh PGP5 (1301 aa) Eh PGP6 (1282 aa) Eh ABC1 (808 aa) Tv PGP1 (589 aa)
2 2 2 2 1 1
Yes ? ? ? ? ?
M88599 M88598 L23922 U01056 U01058 X76160
[39] [39] [37] [37] [131] [41]
PfMDR2 (chr 14)
P. vivax
ABC protein
PfGCN20 (chr 11) gij6594243: (chr 1) 14 884 /20 352d gij3647343: (chr 3) 29 366 /33 463d gij23496770: (chr 12) 185 253 /191 579d gij14578280: (chr 3) 51 639 /55 733d
[28] [50] [50] [31] [35]
N.B. (i) Database searches were performed using the Entrez search and retrieval system for proteins at NCBI. Protein sequences were selected using the program FASTA [133], and analysed for conserved domains using the PSI-BLAST program [134]; (2) the ABC genes LmPGPA (L. major ), and TvPGP2 (T. vaginalis ) have also been identified in parasitic protozoa but to our knowledge no nucleotide sequence accession number has been assigned to any of these sequences so access to these genes and their gene-products was not possible. a Drug resistance mediated by parasitic ABC protein:?, no experimental evidence available; No(?), limited experimental evidence suggests absence of drug resistance. b Only references describing the original characterisation and/or sequencing of the ABC genes are included. c Sequences and annotation of the human malaria parasite P. falciparum are available at the following websites: PlasmoDB (http://plasmodb.org), The Institute for Genomic Research (http://www.tigr.org), the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/Protozoa/), and the Stanford Genome Technology Centre (http://www-sequence.stanford.edu/group/malaria). Chromosome sequences were submitted to EMBL or GenBank with accession numbers AL844501 /AL844509 (chromosomes 1, 3 /9 and 13), AE001362.2 (chromosome 2), AE014185 /AE014187 (chromosomes 10, 11 and 14) and AE014188 (chromosome 12). d Identified by protein BLASTP search (Basic Local Alignment Search Tool for proteins) [132] using as a query the consensus NBD sequence (Accession no. PF00005). The role, if any, of these putative ABC proteins in drug resistance remains to be determined. e Direct submission to GenBank database; LtrMDR1 sequence was submitted by Perez-Victoria, J.M., Gamarro, F. and Castanys, S. (1996); LtrPGPE by Lafuente, E., Castanys, S. and Gamarro, F. (1996).
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305
Fig. 2. Unrooted phylogenetic trees of (A) membrane spanning domains (MD) and (B) NBD identified in ABC proteins of parasitic protozoa. Organisms include P. falciparum (Pf ), E. histolytica (Eh ) and members of the families Trypanosoma (T ) and Leishmania (L ). Sequences from human (h), yeast (y) and C. parvum (Cp ) are included for reference. Sequence labels are defined in Table 2, which also lists accession numbers. Sequences for inclusion were identified using matches found by PSI-BLAST [134,136] http://www.ncbi.nlm.nih.gov/BLAST/) to the consensus sequences for the NBDs (183 amino acids, accession no. PF00005) and MDs (275 amino acids, accession no. PF00664) of ABC proteins. The sequences (gaps removed) were then aligned using ClustalW [137] http://www.ebi.ac.uk/clustalw/) with the default parameters. Distance matrices based on amino acid identity with gaps not taken into account were calculated using TREECON v. 1.3b [138] with Poisson correction. Tree topology was inferred using the neighbour-joining method [139]. Though the trees are unrooted, they are displayed as rooted at the mid-point to make them as compact as possible.
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Fig. 2 (Continued). Bootstrap values are indicated at each vertex. The scale at top left corresponds to 0.1 mutations per position. Shading indicates groups defined by the condition that the greatest distance between any pair of members of the group is less than the smallest distance between any member and any sequence outside the group. In the MD tree there are groups for the N-terminal and C-terminal MD regions of MRP-like sequences. For each of CAB63558 and CpABC the furthest member of the C-terminal group is closer than the nearest sequence not within the group, thus these two sequences should probably be included within the MRP-like C-terminal MD group. In the NBD tree there are three groups corresponding to the N-terminal NBDs of MRP-like sequences, the C-terminal NBDs of MRP-like sequences, and both NBDs of Pgp-like sequences. The N-terminal NBD for CAB 63558 is closer to the furthest member of the N-terminal MRP-like group than to any other sequence in the table (except CpABC 2 which is roughly equidistant). Thus, the NBD of CAB63558 should probably be within the MRP-like N-terminal NBD group.
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Fig. 3. Predicted topology of ABC-type transporters in parasitic protozoa. (A) Pgp cluster. Members of this cluster contain two MDs (MD1 and MD2), each consisting of six a-helical TMS, and two NBDs (NBD1 and NBD2). The individual domains are fused into a single multifunctional polypeptide with the {MD-NBD}2 topological configuration. (B) TAP cluster. The typical ABC transporter of this cluster contains a single MD fused to a single NBD. These transporters are known as ‘‘half-size’’ transporters with the {MD-NBD} topology (a). Variations of such a configuration include the {MD}2-NBD topology (b). Proteins with this topology contain a non-conserved N-terminal MD (MD1) (see text). (C) MRP cluster. Typical members of this cluster also have the {MD-NBD}2 topology (a) but they contain a long hydrophilic N-terminus, diagnostic for MRP members. Atypical members of this cluster show the {NBD}2 topology (b) (see text). Representative members of each cluster are mentioned in the figure. The predicted topology of the hypothetical proteins identified in parasitic protozoa and that of the Eh ABC and Tb ABC2-3 proteins has not been included in this figure because the prediction of their membrane-associated domains has not been conclusive.* C-terminal NBD missing because of a retrotransposon.
In T. brucei , three partially cloned ABC genes designated TbABC1 , TbABC2 , and TbABC3, containing NBD sequences, were initially identified by PCR techniques [42]. TbABC2 and TbABC3 do not at present clearly fall into either the Pgp or the MRP cluster (Fig. 2). Cloning of the complete cDNA sequence for TbABC2 and TbABC3, and their functional analysis has not yet been reported. On the other hand, the sequence of TbABC1 has now been identified to be identical to the NBD2 sequence of the recently cloned and characterised MRP homologue TbMRPA in T. brucei [43]. The first Pgp homologue identified in P. falciparum is located on chromosome 5 of P. falciparum and encodes a 162-kDa protein, termed P-glycoprotein homologue 1 (Pgh1) or Pf MDR1 [26,27]. The gene is expressed in both drug-sensitive and drug-resistant P. falciparum strains in the form of two transcripts. One of these transcripts (8.5 kb long) is present at all asexual
erythrocytic stages [26], whereas the other transcript (7.5 kb long) is only present during the asexual trophozoitic stage [44]. The PfMDR1 protein exhibits overall 33% amino acid identity with human Pgp [12] and has an {MD-NBD}2 topology (Fig. 3A). The second putative Pgp homologue identified in P. falciparum is located on chromosome 14 of P. falciparum and encodes a 110-kDa protein, termed Pf MDR2 or P-glycoprotein homologue 2 (Pgh2) [45 / 47]. The domain organisation of PfMDR2 protein is rather unusual with ten putative TMS (four in MD1 and six in MD2) and a single C-terminal NBD (Fig. 3B). It is not yet known whether MD1 and MD2 form a single extended MD or whether they represent two separate MDs. MD2 exhibits a significant sequence conservation with the MD of the heavy metal tolerance protein (HMT) in the fission yeast Schizosaccharomyces pombe [48,49]. HMT is a typical half-Pgp transporter with an {MD-NBD} topology.
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4. Protozoal ABC transporters included in the MRP cluster In Leishmania , the first MRP homologue described was LtPGPA [50]. This gene was discovered on an extrachromosomal circle (H-circle) amplified in a methotrexate-resistant Leishmania tarentolae promastigote cell line [28]. LtPGPA gene is also amplified in cell lines resistant to terbinafine, primaquine, and arsenite [51 / 53]. A plasmid-encoded LtPGPA homologue in L. major, LmPGPA , was shown to confer resistance to heavy metal ions [29]. LtPGPA is a member of a multigene family in L. tarentolae that includes four other family members, LtPGPB to LtPGPE [50]. A putative homologue of the LtPGPE gene, LtrPGPE , has also been described in methotrexate-resistant L. tropica cell lines [54]. Analysis of the phylogenetic trees confirms that the Lt PGPA, Lt PGPB, Lt PGPE, and Ltr PGPE proteins are all placed within the MRP cluster (Fig. 2). Interestingly, the Leishmania MRP homologues all have a {MD-NBD}2 topology, similar to that of the Leishmania Pgp homologues (Fig. 3C). Databases search using BLASTP has identified a putative MRP homologues in L. major , termed CAB64568 (Table 2, Fig. 2). Analysis of the predicted protein sequence shows that CAB64568 protein has the same {MD-NBD}2 topology as mentioned above. MRP homologues have also been studied in Cryptosporidium parvum , a protozoan parasite that causes an important enteric disease known as cryptosporidiosis. The parasite develops inside a vacuole at the apex of its epithelial host cell and is separated from the host cell cytoplasm by an extensively folded membrane structure known as the feeder organelle. The feeder organelle is suggested to be the site of regulation of transport of nutrients and drugs into the parasite. An ABC transporter gene, termed CpABC , encoding a putative 162kDa ABC protein has been localised to the host/ parasite boundary, possibly the feeder organelle [55 / 57]. The predicted amino acid sequence of Cp ABC shares a significant similarity with that of the cystic fibrosis conductance regulator (CFTR), a chloride channel [58], and the multidrug resistance-associated protein 1 (MRP1) [56]. Several MRP homologues have been described in T. brucei , the causative agent of African sleeping sickness and animal trypanosomiasis. The most recently discovered ABC genes in T. brucei are TbMRPA and TbMRPE [43]. Overexpression of TbMRPA and TbMRPE in T. brucei resulted in resistance to melarsoprol and suramin, respectively. Amino acid sequence analysis suggests that Tb MRPA is most closely related to LtPGPA and Tb MRPE to Lt PGPE. Both the proteins exhibit the typical Leishmania MRP topology, {MD-NBD}2. ABC transporter genes, termed TcPGP2 and TcPGP1A , have also been described in T. cruzi , the
causative agent of South-American Chagas’ disease [59,60]. Both genes encode ABC proteins that are homologous to the MRP homologues Lt PGPA/B and Ltr PGPE in Leishmania (Fig. 2). Surprisingly, the TcPGP1A gene seems to lack the DNA sequence encoding the C-terminal NBD due to the insertion of a retrotransposon [60]. Gene transfection experiments with the T. cruzi MRP homologue, TcPGP2, in L. tropica suggested that the Tc PGP2 protein is not involved in drug resistance [59]. MRP homologues are probably also expressed in P. falciparum . Our database search has identified open reading frame CAB63558 on chromosome 1 of P. falciparum strain 3D7, which encodes a putative protein of 1822 amino acids containing two NBDs and two MDs in an {MD-NBD}2 topological configuration. CAB63558 shares significant sequence identity with Arabidopsis thaliana MRP2 and C. parvum Cp ABC. In addition, an open reading frame on chromosome 11 of P. falciparum , termed PfGCN20 , encodes an ABC protein that shares partial homology with mammalian MRPs [61]. Pf GCN20 also shares homology with the yeast translational regulator protein, GCN20P (Fig. 2B) [61]. The predicted protein structure of Pf GCN20 shows that this protein contains two NBDs, but no MDs (Fig. 3C).
5. ABC transporters and multidrug resistance in Leishmania and Trypanosoma Drug resistance is a major obstacle to the success of chemotherapy against leishmaniasis and trypanosomiasis because the development of new drugs is proceeding very slowly and vaccines are not yet available [62 /65]. The drugs of choice for treating all forms of leishmaniasis, ranging from self-healing cutaneous lesions (oriental sore) to fatal visceral infection (kala-azar), remain the pentavalent antimonial compounds (SbV) despite their renal and cardiac toxicity. Melarsoprol, the only drug for the late stage sleeping sickness, has proven to be less effective because of the global development of drug resistance in the parasite [13]. 5.1. PGPA and PGPE in Leishmania PGPA and PGPE have been implicated in the resistance of Leishmania to the metal oxyanions antimony and arsenite [8,9,11]. Expression of Lt PGPA in transfected L. tarentolae resulted in a low level of resistance (2-fold increase) to oxyanions such as arsenite and pentavalent antimonials [66]. However, similar experiments in L. major with LmPGPA resulted in a 10-fold higher level of resistance to arsenite and trivalent antimonials, but not to pentavalent antimonials, zinc, cadmium, or to the cytotoxic drugs vinblastine and
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puromycin [29]. The different resistance levels described between LtPGPA and LmPGPA were clearly dependent on which PGPA allele was used and in which Leishmania species the genes were transfected. In drug resistant Leishmania cell lines, the overexpression of LtPGPA has been attributed to the increased copy number of the H-plasmid that harbours LtPGPA [50]. Extrachromosomal gene amplification is a common mechanism of gene overexpression in drug resistant Leishmania parasites, which may explain in part why so many amplified ABC genes have been observed in drug resistant mutants [67,68]. Intrachromosomal amplification is rare in Leishmania but frequent in Plasmodium where the size of the chromosome is much bigger [69]. The observation, that resistance levels to metal oxyanions were dependent on which PGPA allele was used and in which Leishmania species the genes were transfected, suggested a role of additional factors or genes in high levels of resistance to metal oxyanions [8,70/73]. Given the structural similarity of Lt PGPA to MRPs, it was suggested that LtPGPA may mediate the active efflux of metals as a metal-thiol complex, similar to observations for mammalian MRP 1 and 2, and hence, that the intracellular thiol concentrations in Leishmania would be an important factor in metal oxyanion resistance of the organism [8,74]. Soon after this suggestion, transport studies provided solid evidence that Lt PGPA transports metals when conjugated to thiols such as reduced glutathione (GSH) [75] and trypanothione (TSH) [71,76]. TSH consists of a spermidine moiety linked to two GSH molecules [77]. GSH is the tripeptide g-Glu /Cys /Gly that plays an important role in the cellular defence against oxidative stress and in the detoxification of several drugs and xenobiotics [78]. Consistent with the role of TSH and GSH in oxyanion resistance in Leishmania , a large increase in TSH and smaller increases in cysteine and GSH were observed in all metal resistant L. tarentolae analysed [71,73,79]. The mechanism that led to increased levels of TSH was shown to differ between various Leishmania species. In L. tarentolae this increase was due to amplification of GSH1 , encoding g-glutamylcysteine synthetase (a key enzyme in glutathione biosynthesis), whereas in L. tropica and L. maxicana GSH1 was not amplified [79]. In addition to the upregulation of glutathione biosynthesis in oxyanion resistant L. tarentolae, the biosynthesis of spermidine was also upregulated through overexpression of the ODC gene encoding ornithine decarboxylase [73]. It was also shown that GSH1 , ODC and LtPGPA act synergistically to confer high-level resistance to metals when co-transfected into Leishmania [70,72]. The above data have led to the proposal that high levels of PGPA-mediated oxyanion resistance in Leishmania are due to a combination of elevated concentrations of trypanothione and an increased metal-thiol transport activity [11]. The localisation of
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Lt PGPA in intracellular compartments rather than in the plasma membrane of L. tarentolae suggests that this transporter confers resistance by sequestering the metalthiol conjugates into intracellular vesicles [80,81]. 5.2. MRPA and MRPE in Trypanosoma Two ABC transporters, Tb MRPA and Tb MRPE, are involved in drug resistance of Trypanosoma to antitrypanosomal drugs. Overexpression of TbMRPA , a homologue of LtPGPA gene, in T. brucei resulted in a high level of resistance (10-fold) to melarsoprol and a lower level of resistance to berenil. However, trypanosoma cell lines overexpressing Tb MRPA remained sensitive to suramin and pentamidin. Hence, the resistance of trypanosomes to suramin and pentamidin must be based on other transporters or alternative mechanisms of drug resistance. Subsequent studies showed that overexpression of the Lt PGPE homologue TbMRPE in T. brucei correlated with significant resistance to suramin (3-fold), marginal resistance to melarsoprol, and sensitivity to pentamidin suggesting that suramin and melarsoprol are both transported by Tb MRPE [43]. Similar to Leishmania , the drug resistance in trypanosomes is based on a trypanothione and glutathionedependent conjugation of drugs, followed by the transport of the drug conjugates. However, in contrast to the L. tarentolae in which levels of trypanothione and glutathione were increased in in vitro selected cell line resistant for arsenical drugs [71], the levels of trypanothione or glutathione in T. brucei were unaltered in melarsen resistant cell lines [82]. Further increase in the levels of trypanothione and glutathione in T. brucei by overexpression of ODC/GCS only slightly increased Tb MRPA-mediated melarsen resistance [83]. Taken together, these results suggest that in T. brucei , the endogenous level of glutathione/trypanothione is sufficient for conjugation of melarsen and other drugs. Immunfluorescence studies suggest that Tb MRPA is localised on the plasma membrane of the parasites, indicating the protein confers resistance by transporting the drug out of the cell. In contrast, Tb MRPE is localised in an intracellular organelle between the nucleus and kinetoplast, and hence, this protein may transport drugs into this organelle [43]. 5.3. Leishmania MDR1 The mechanism by which Leishmania MDR1 confers drug resistance is unclear. The MDR1 gene is amplified in several Leishmania species selected for resistance against substrates of the mammalian Pgp MDR1 (e.g. daunorubicin, vinblastine), and it was suggested that this amplification is the cause of the increased resistance to these drugs [32 /35]. Other mechanisms for the upregulation of the expression of Leishmania MDR1 have
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also been proposed. Recently, it was hypothesised that polymorphism in the 3? untranslated region of the MDR1 gene in drug resistant L. enrietti may increase RNA stability and enhance Le MDR1 synthesis [84]. This hypothesis is now under investigation. Interestingly, the Leishmania MDR1 localises in subcellular organelles, although the exact location remains to be determined. It was originally suggested that the Leishmania MDR1 is localised in intracellular vesicles close to the mitochondria but recent evidence suggests that this might not be the case. A novel vesicular structure, the multivesicular tubule (MVT) is believed to be involved in exocytosis and endocytosis in Leishmania , and has been suggested to harbour the MDR1 protein in L. enrietti [84]. Irrespective of the precise intracellular location of the Leishmania MDR1, the subcellular location of this transporter suggests that drug resistance in this parasite may be mediated by mechanisms that are different from the conventional efflux mechanism across the plasma membrane, as observed for mammalian Pgp MDR1. Intracellular expression of the Leishmania MDR1 indicates that one possible mechanism of drug resistance involves the separation of drug from its target of action (sequestration model) [85]. The plasmodial ABC transporter, Pf MDR1, has been suggested to confer resistance by a similar sequestration model [85]
6. ABC transporters and multidrug resistance in Plasmodium falciparum Chemotherapeutic treatment of infections by P. falciparum has relied primarily on quinoline-containing drugs (quinine, CQ, mefloquine) but also on halofantrine, folate antagonists, and to a lesser extent on tetracycline and derivatives of artemisinine (Table 3) [86]. Prior to the development of drug resistance CQ, a 4-aminoquinoline diprotic weak base, was the drug of choice for treating malaria. Quinolines inhibit polymerisation of the toxic oxidised haem (haematin) that is released during haemoglobin degradation within the digestive food vacuole of the parasite (Fig. 4) [87 /94]. However, P. falciparum has developed CQ resistance, which often coincides with resistance to other antimalarials such as mefloquine, artemisinin and halofantrine [95 /98] and this resistance is now widespread in every geographic region where malaria is endemic [99]. Early studies linked antimalarial resistance of P. falciparum to a reduction in drug accumulation, which was suggested to result from reduced drug uptake [92], enhanced drug efflux [25], and/or modulation of DpH across the food vacuole membrane [100]. Since the cloning and initial characterisation of PfMDR1, impressive progress has been made in understanding the mechanism(s) of antimalarial drug resistance and the
Table 3 Major antimalarial drugs in use Generic chemical group
Examples
Antifolic drugs Sulpha drugs
Pyrimethamine, Proguanil (a) Sulphones, e.g. Dapsone (b) Sulphonamides, e.g. Sulphadoxine Cinchona alkaloids Quinine 4-Aminoquinolines Chloroquine, Amodiaquine 8-Aminoquinolines Primaquine 4-Quinoline methanol Mefloquine 9-Phenanthrene Halofantrine methanol Sesquiterpene lac(a) Artemisinin tones (b) Artemisinin derivatives e.g. Artemether Antibiotics Tetracycline, Chloramphenicol Drug combinations Pyrimethamine/Sulphadoxine (Fansidar) Pyrimethamine/Sulphadoxine/Mefloquine (Fansimef) Atovaquone/Proguanil (Malarone)
involvement of the Pf MDR1 transporter in the resistance processes. However, contradictory and sometimes confusing data have raised considerable debate around two main issues: (i) the role of Pf MDR1 in antimalarial drug resistance in P. falciparum , and (ii) the role of Pf MDR1 in CQ efflux. The relationship between Pf MDR1 expression and drug resistance is complex. Early findings suggested a link between PfMDR1 amplification, Pf MDR1 overexpression, and CQ resistance in the parasite [26,101]. Later findings suggested that PfMDR1 expression levels do not vary between CQ-sensitive and CQ-resistant parasites for certain strains, and that hence, Pf MDR1 overexpression was not essential for CQ resistance [102]. In contrast, there is consistent evidence for amplification of the PfMDR1 gene in mefloquine-resistant P. falciparum in laboratory and field isolates [27,44,103,104]. Interestingly, some studies have shown that the copy number and expression of the PfMDR1 gene are inversely correlated with CQ resistance but positively correlated with mefloquine resistance [105 /107] with parasites selected for CQ resistance showing reduced amplification of PfMDR1 and decreased resistance to mefloquine [105]. In contrast, parasite selected for mefloquine resistance exhibited amplification of PfMDR1 and decreased resistance to CQ [106,107]. Selection for mefloquine resistance also resulted in cross-resistance to other antimalarials, including halofantrine and quinine [103,106,107]. Recent studies have shed further light on the involvement of PfMDR1 in antimalarial resistance [108,109]. Three polymorphic mutations (S1034C, N1042D, and D1246Y) of PfMDR1 previously identified and shown to enhance CQ resistance [101] were stably transfected into CQ-sensitive parasites containing the wild-type
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Fig. 4. Proposed transport activities in the P. falciparum digestive vacuole. The Plasmodium digestive food vacuole is an acidic proteolytic compartment (pH /5), important to the metabolism of the parasite. Haemoglobin, which serves as a major source of amino acids, is pinocytosed into the parasite in vesicles that fuse with the food vacuole. A proton ATPase pump is responsible for vacuole acidification, which is required for activity of the proteases that release globin fragments and toxic haem (haematin). The globin fragments are digested further into small peptides, which are probably transported out of the vacuole before being converted into free amino acids in the parasite cytoplasm. The haem is partly detoxified by a polymerisation process, which results in the formation of an inert crystal called malarial pigment or haemozoin. The iron of the haem becomes oxidised with formation of reactive oxygen species. These are detoxified either by superoxide dismutase (SOD) and catalase or by glutathione peroxidase. If exposed to quinolines these accumulate to high levels within the vacuole, and inhibit haem polymerisation, causing a buildup of free haematin or haematin/quinoline complex that ultimately kills the parasite. Pf MDR1 has been localised to the food vacuole but controversy exists as to whether it is able to transport into the vacuole as shown at the bottom of the figure for chloroquine (CQ) or out, as shown alternatively for CQ and for mefloquine (MQ) and for small peptides. A role in transport of peptides is plausible given the ability of this protein to transport the yeast mating pheromone a-factor (12 amino-acid peptide) [110]. Active efflux of MQ and CQ out of the food vacuole in resistant strains of Plasmodium, if it were to be mediated by Pf MDR1 would provide a plausible explanation for association of Pf MDR1 with resistance. However, there is also evidence that Pf MDR1 may mediate influx of CQ into the food vacuole as expected if Pf MDR1 transports by the same mechanism as mammalian Pgp, i.e. away from the cytoplasmic side containing ATP. Compiled from [108,140 /142].
PfMDR1 gene [108]. Expression of the mutant Pf MDR1 proteins conferred resistance to quinine, and as expected, to CQ (in a strain-specific manner) but not to mefloquine, halofantrine and the structurally unrelated antimalarial artemisinin [108,109]. Collectively, the findings that (i) Pf MDR1 and mammalian Pgps are homologous proteins, (ii) amplification of wild-type PfMDR1 results in resistance to mefloquine and structurally unrelated drugs and (iii) changes in the amino acid sequence of PfMDR1 affect drug specificity, support the notion that Pf MDR1 plays an active role in multidrug resistance in P. falciparum . However, the question remains by which mechanism Pf MDR1 mediates drug resistance. The majority of studies on mechanisms have focused on CQ resistance as this has been the main antimalarial drug. Protein localisation studies have indicated that Pf MDR1 is primarily expressed in the membrane of the digestive food vacuole and to a lesser extent in the plasma membrane, suggesting that this protein has a possible physiological function in subcellular organelles [102].
The first clear evidence supporting the functional role of Pf MDR1 as a drug transporter came from the ability of Pf MDR1 to functionally substitute for the transport activity of the yeast mating a-factor efflux pump STE6 in the STE6-deficient S. cerevisiae SM1563 [110]. In addition, expression of a mutant PfMDR1 gene containing two naturally occurring polymorphisms that are associated with choloroquine resistance (N1042D and S1034C) [101] abolished the functional complementation of a-factor transport in S. cerevisiae SM1563 [110]. Taken together, these data argue that PfMDR1 can act as an efflux pump and that the mutations in Pf MDR1 that are associated with CQ resistance alter this ability. It is still unclear what mechanisms bring about CQ resistance in P. falciparum . Decreased accumulation of CQ within the food vacuole of the parasite has been observed (Fig. 4) [111,112] but whether or not these events are brought about directly by changes in Pf MDR1 mediated transport is uncertain. Older ideas (model A, Fig. 5) were that reduced accumulation of CQ was the result of PfMDR1-mediated CQ export from
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the food vacuole [25,113]. Others have suggested (model B, Fig. 5) that this was the result of a decrease in Pf MDR1-mediated import of CQ into the food vacuole. Though model A has been criticised there is a considerable body of evidence to suggest that polymorphic mutations in the primary structure of Pf MDR1 do affect CQ efflux from the food vacuole [108,114]. It is more difficult to reconcile the events in model B with the evidence that inhibition of Pf MDR1 activity by inhibitors (such as verapamil) results in increased accumulation of CQ in the food vacuole. Indeed there are two Pf MDR1-independent models that may explain the decreased accumulation of CQ in the food vacuole of drug resistant parasites. In model C (Fig. 6), drug resistance is thought to be due to alkalisation of the parasite’s food vacuole, and hence, a reduced trapping of CQ in the organelle. In model D (Fig. 6), CQ resistance is related to enhanced activity of a plasmodial Na /H exchanger (Pf NHE) which, by expelling protons in exchange for sodium ions across the plasma membrane, would result in an alkalisation of parasite’s cytoplasm with a concomitant reduced accumulation of CQ in both cytoplasm and the food vacuole. Interestingly, even though Pf MDR1 may contribute to CQ resistance in certain P. falciparum strains [26,101], the region on chromosome 5 encoding Pf MDR1 does not necessarily segregate with CQ resistance in the progeny of a genetic cross between CQ-resistant (Dd2) and CQ-sensitive (HB3) P. falciparum parasites [115 /117]. Re-examination of the locus on chromosome 7 that segregated with CQ resistance in the genetic cross pointed to a role of the novel 13-exon
polymorphic gene PfCRT in CQ resistance [118]. Consistent with this notion, mutations at codons 76 220 and others in the PfCRT gene appeared to be the central determinant for CQ resistance in P. falciparum [119 /123]. Pf CRT is a polytopic membrane protein, but not an ABC transporter, and is localised in the membrane of the food vacuole. The activity and physiological role of this important protein remain to be established. The partial reversal of CQ resistance by verapamil suggests that the CQ resistance phenotype is based on multiple factors/components: one verapamilsensitive component mediated by Pf MDR1, and other verapamil-insensitive component(s) such as Pf CRT (see below) [101,108,117,118,124]. The observation that certain PfMDR1 mutant genes (e.g. N86Y) can enhance the level of resistance related to PfCRT expression with a K76T mutation in Sudanese isolates of P. falciparum suggest a joint action of the two genes in high-level CQ resistance [125]. The understanding of the genetics of CQ resistance in P. falciparum is, however, by no means complete. For example, Chen [126] and colleagues have provided evidence that, in addition to PfCRT and PfMDR1 , other genetic loci may be involved in the modulation of CQ-resistance levels in P. falciparum in Thailand, supporting the early suggestion that the CQ-resistant phenotype is multigenic and that mechanisms on loci other than PfCRT and PfMDR1 are likely to modulate the level of CQ resistance [108]. It seems thus that ABC transporters may be acting alongside other proteins to bring about drug resistance.
Fig. 5. Models for the involvement of Pf MDR1 in CQ resistance. The figure shows a traditional view of the malaria-infected erythrocyte, in which solute and drug trafficking between the parasite and the extracellular medium occurs via the erythrocyte cytosol, crossing the red blood cell membrane (RBCM), the parasitophorous vacuole membrane (PVM), and the parasite plasma membrane (PPM). In Model A, Pf MDR1 on the food vacuole (FV) mediates the active efflux of CQ from this organelle. In Model B, Pf MDR1 mediates the active import of CQ into the FV, impairment of which causes decreased CQ accumulation in this organelle.
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Fig. 6. Models for the involvement of membrane transport processes in CQ resistance. In Model C, an increased vacuolar pH arising from (i) the reduced activity of the V-type H ATPase and/or increased H leakage, or (ii) impaired Cl conductance in the FV, imposes a limitation on the electrogenic flux of CQ into the vacuole, thus decreasing the CQ concentration in this organelle. In Model D, the influx of CQ across the PPM via an unknown mechanism is mediated by a plasmodial Na /H exchanger (Pf NHE), and limited by altered properties of this exchanger.
With regard to other ABC transporter homologues of mammalian Pgps and MRPs, in P. falciparum there is no clear evidence as yet to link any of these with plasmodial drug resistance. This conclusion includes PfMDR2 [45,47]. The protein encoded by this gene is localised in the plasma membrane [47] and food vacuole membrane [45] throughout the asexual intra-erythrocytic life cycle of the parasite but its biological function remains unknown though its structural similarity to HMT may suggest a potential role of the protein in plasmodial metal homeostasis. Another plasmodial protein containing the ABC signature and identified in the MRP cluster, Pf GCN20, has not yet been linked to drug resistance since no differences are observed in expression level in either CQ or mefloquine resistant and sensitive strains [127].
7. Conclusions The past 5 years has seen a pronounced re-awakening of interest in parasitic diseases. At present, a multiplicity of resistance mechanisms has been detected, the most prevalent ones being transporter mutations and amplification of transporter genes. In many cases, e.g. the PGPA-mediated oxyanion resistance in Leishmania , the role of ABC transporters in drug resistance is well established. In addition, the impressive progress in understanding of the CQ resistance mechanisms in P. falciparum and the very substantial literature on the accumulation of CQ by malaria-infected erythrocytes has shown that transporters belonging to the ABC superfamily and other families are relevant. The pub-
lished genome sequences of P. falciparum [128] and Plamodium yoelii yoelii [129] and the ongoing genome sequence projects for other parasitic protozoa will reveal a wealth of information about their physiology and pathogenicity, and will initiate many new biochemical studies on the structure and function of parasitic transport proteins involved in multidrug resistance and other cellular functions.
Acknowledgements We would like to thank Dr Lekshmy Balakrishnan and Dr Henrietta Venter for proof-reading the final version of the manuscript. Research of the Barrand and Hladky group is funded by the Sir Jules Thorn Charitable Trust, the Biotechnology and Biological Sciences Research Council (BBSRC) and Cancer Research UK. Research of the VanVeen group is funded by the Association of International Cancer Research (AICR), BBSRC, Cancer Researchf UK, Medical Research Council (MRC), Royal Society, and Molecular Devices Ltd.
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